Photoperiod-driven developmental and metabolic remodeling supports pre-diapause energy reserve accumulation in Hyphantria cunea.

The fall webworm, Hyphantria cunea, is a destructive invasive pest whose seasonal outbreaks depend largely on successful pupal diapause, but how photoperiod coordinates larval development and fat body transcription during pre-diapause preparation remains incompletely understood. Here, we characterized photoperiod-associated developmental and transcriptional remodeling before diapause and assessed candidate genes linked to diapause-related traits. Under short photoperiod, diapause-destined (DP) larvae developed an additional seventh instar. Relative to non-diapause (NDP) larvae under long photoperiod, they exhibited enlarged fat bodies, larger pupae, and greater terminal-stage body mass, consistent with enhanced storage capacity before pupation. Fat body transcriptomics across matched fourth- to sixth-instar comparisons yielded 4154 differential-expression calls, and functional profiling highlighted stage-specific metabolism- and hormone-related processes. Independent RT-qPCR analysis confirmed the RNA-sequencing expression patterns. Hc_CYP314A1 and Hc_Wat showed their strongest DP-biased expression at the sixth instar. Under diapause-inducing conditions, feeding-based RNA interference linked eight candidate genes to gene- and sex-dependent changes in developmental timing, survival, pupal weight, pharate-adult development, and eclosion timing. The dsDP_RFP control eclosed only after prolonged diapause, whereas Hc_Diap1-4 knockdown produced earlier eclosion in subsets of pupae at 0-30 or 90-110 d. Together, these findings reveal coordinated photoperiod-associated developmental and fat body transcriptional remodeling during pre-diapause preparation and identify candidate genes associated with diapause-related traits in H. cunea.